JPH0272899A - Method for detecting a substance having a hydrolase activity and a bicyclic compound - Google Patents

Abstract

The invention relates to a method for detecting substances with hydrolase activity in a sample by mixing the sample with a hydrolase substrate and with an oxidising agent and evaluating the resulting colour intensity, characterised in that the hydrolase substrate is at least one compound of the formula I <IMAGE> in which R<1> is hydrogen or an alkoxy group R<2> is hydrogen, halogen, an alkoxy, an aralkoxy or amino group R<3>, R<4>, R<5> and R<6>, which can be identical or different, are hydrogen, halogen, an alkyl, alkoxy, aralkoxy, carboxyl, alkylcarbonyl, alkoxycarbonyl, carbamoyl, sulpho or amino group and X is a glycosyl, phosphate or acyl radical, where the radicals R<2> and R<3>, R<3> and R<4> and R<4> and R<5>, and R<5> and R<6> can each form a closed ring system, at least one of the radicals R<1> and R<2> is hydrogen and at least one of the radicals R<1>, R<2> and R<3> is not hydrogen, to a means for carrying it out and to hydrolase substrates.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The objects of the present invention are a method for detecting a substance having hydrolase activity, a drug suitable for the detection, a novel hydrolyzed arsenic substrate, and a method for producing the same.

[Conventional technology]

Hydrolase is an enzyme that has recently become very important. On the one hand, this enzyme is found in plants,
It plays an important role in the metabolism of animals and also humans. Disease can occur if the concentration of hydrolytic enzymes in one of these biological systems differs from the concentration normally present there. Therefore, when a disease is present, the ek of hydrolytic enzymes in body fluids is
One of the challenges of clinical diagnosis is to determine the difference between cases and standard values by measuring the degree of irradiation. This is advantageously carried out using the determination of hydrolytic enzymes by indicator reaction. For this purpose, a substrate for the relevant hydrolytic enzyme is added to the sample to be tested. It is a measure of the concentration of hydrolytic enzymes at which the product produced from the substrate is present.

In Ikekata, hydrolytic enzymes are often used as enzymes for labeling immunologically active compounds. At that time, β-
However, the lactosyl group is particularly widely used [Annals
Ode Clinical Biochemistry (Annals)
ofClinical Biochemistry
) K vol. 16, p. 221 (1979)]. The test in this article is useful for determining the analyte content of immunological activity in a sample. These measure the degree of analyte using a compatible immune partner with covalently attached β-lactosidase as a label. By carrying out the test, the concentration of the immune partner is matched to the concentration of the analyte to be measured! Depends on the relationship. Labeled immune partner molecules are likewise rendered visible by indicator reactions. In an indicator reaction, the immune partner labeled with β-glycosidase is reacted with the β-glycosidase group.

The amount of product produced is proportional to the concentration of the immune partner.

Tests prepared using samples with known analyte complexes
By comparing to the value of the line, the unknown analyte concentration in the sample can be determined.

Hydrolase is a method for detecting nucleic acids.
It is also used as an enzyme for identification. A method of this type using β-glycosidase as an enzyme label is described in DE-A 2915082. Even in this case, the labeled iI is measured by indicator reaction.

Hydrolytic enzymes are also used as trace reagents.

Again, it is important to measure the enzyme concentration accurately and quickly. For this purpose, methods similar to those commonly used in clinical diagnostics are used.

In this case, the substrate is usually a compound that is soluble in the sample liquid. Upon reaction with a hydrolytic enzyme, a product is produced that differs from the substrate in one of its properties, such as a characteristic light absorption, light emission (fluorescence), etc., and can thereby be detected.

Test methods based on immunologically active substances are divided into two technical aspects. In one frequently used embodiment, the detection reaction is performed in a cuvette such as those commonly used in photometric testing. Evaluation of the indicator reaction is carried out by measuring absorption, luminescence or radioactivity with suitable equipment.

In another embodiment, the reaction is carried out in one or several layers of a fleece or film that is part of the test indicator. The necessary reagents are applied onto these fleeces or films. The t' of the products liberated by the hydrolase activity from the hydrolase substrate during the indicator reaction can be directly measured in fleeces or films of this type.

One of the advantages of this embodiment is that only a single solution of the mostly liquid sample can be operated using sound, which greatly simplifies the implementation of the method. In particular, the product deposits produced can be easily tracked by measuring the absorption of light at specific wavelengths.

A suitable substrate for this is European Patent (GP-A) No. 01
Phenolsulfonephthaleinyl-β-D-, which is described in the specification of No. 46866, is lactocyto and its derivatives, from which the phenolsulfonephthalein derivative is liberated by the reaction of β-ri with lactondase. The substrate has a yellow coloration while the product has a red coloration. During the indicator reaction, a gradual color change occurs from yellow to orange to red. Since this color change can only be evaluated very inaccurately with the naked eye, a suitable photometer is used for measurement.

Resorufin-β-D-glycoside described in European Patent (EP-A) No. 0156347 corresponds to a substrate that undergoes a color change from yellow to red upon reaction with glycosidase.

Since it has been found that the visual evaluation leads to subjective results, especially at low β-glycosidase activities in the yellow to red transition range, a device is used for the evaluation here as well. However, these devices are relatively complex and therefore expensive. Torfin glycosides are not suitable for use as substrates in test supports.

It is generated from them and the torphin bleeds (ausbruten).

Hydrolytic enzyme substrates, such as methylumbelliferone derivatives, also undergo only a change in fluorescence upon hydrolysis and are likewise not suitable for visual evaluation. These also showed deIJ-Pl on the specimen.

Produces easy-to-understand substances. For example, methylumbelliferone, which is produced from lactone r, is easily deleted.

(In 1999, a method for detecting substances with hydrolase activity was proposed that uses uncolored hydrolases that convert into colored products upon reaction with hydrolases. Therefore, these detection reactions do not involve color change, but , the formation of pigments is evaluated completely.This is the color intensity of the color scale CF'arbenskal.
This can be easily done by comparing the color tone with a).

This type of test can also be used to evaluate people who are color blind.

A substrate of this type is the 5-bromo-4-chloro-6-indolyl-β-lactone f. This substrate produces the color "X" after decomposition and oxidation, but the dye is deleted on the test support.However, the substrate itself is only slightly soluble in water;
β- is only slowly eliminated from the lactondase.

The intensity of the coloring that occurs is therefore only very low. Therefore, tests based on this are also not suitable for rapid visual evaluation.

Bioch
emische Zeitschrift) No. 665
In Vol. 209i (1960), a compound from which phenol is liberated by hydrolysis was proposed as a substrate. However, it is easy to delete on the test support. In the case of nitrophenol, a more or less intense yellow coloration occurs during the indicator reaction. Visual evaluation is very disadvantageous in a small concentration range at this wavelength.

For better visibility, the phenol liberated in an additional reaction may be oxidatively combined with aminoantipyrine or with methylben-dothia-nonhydra.
Analytical Biochemistry
ical Biochemistry) Volume 40, 2
81 (1971)] or reacted with a diazonium salt to turn it into an a-do dye [for example, histohyemy() (i
stochemie) Volume 37, page 89 (197ri 1
Year)〕. Many of these dyes are not very easy to delete and are therefore certainly suitable for use in test supports; however, the use of these coupling reactions is disadvantageous for other reasons. In order to carry out the test on a fleece or film, all the reagents involved in this additional reaction must also be coated on the fleece or placed in the film. These large numbers of reagents, e.g. coupling components, complicate the assay @ implementation and are necessarily associated with a number of drawbacks and cone points; therefore, in individual cases it is necessary to prevent the reagents from prematurely warping against each other or other components of the sample solution. Care must be taken to ensure that it does not react in a non-specific manner with other ingredients.

That is, aminoantipyrine or methylbenzothia alinone hyzolatine may also react with, for example, components in the test bed, leading to erroneous measurements. More cups! ) ingredients, such as shea and tonium salts, impair storage stability. Even when selecting all reagents, consideration must be given to the fact that the reagents themselves already have all their own unique colors.

Delete phenomena that occur on the test support with some of the pigments formed will, for example, cause the color intensity to not be uniform in all locations of the fleece or film, which may be a drawback during evaluation. This means, for example, that the measured values will be incorrect.

[Problem to be solved by the invention]

Therefore, there was a need for a method that would eliminate the above-mentioned drawbacks and would allow substances with hydrolase activity to be detected easily, quickly and reliably even on a test support.

The problem of the present invention was to meet this need. [Problem 't - Means for Solving] This problem was achieved by mixing a sample with a hydrolase substrate and an oxidizing agent and evaluating the resulting color intensity. The problem is solved by a method of detecting a substance having hydrolase activity in a sample, which uses as a hydrolase substrate a compound of the formula I: [wherein R1 represents a water cloud or an alkoxy group, R''h water R, Halogen, alkyl-aralkoxy- or amino-based radical R3H4, R5 and R6 may be the same or different, hydrogen, halogen, alkyl-alkyl, aralkoxy-carzasilyl, alkylcarbinyl, alkylcarbinyl- represents a carbamoyl-, sulfo- or amino-group, and X represents a glycosyl-, phosphate- or acyl group;
r5 and R4 and R4 and R5 and R6 and R6 may each be closed to form a ring system, at least one of the groups R1 and R2 represents a water gun, and at least one of the groups RI R2 and R3 represents a water cloud. at least I
N! It is characterized by using f.

The alkoxy radicals of the radicals RI R2R3R4R5 and R6 advantageously consist of 1 to 4 carbon atoms, which may form a single 44 or be branched. Particular preference is given to methoxy-inpropoxy-n-propoxy- and n-
-butoxy group.

The alkoxy groups of the groups RI R2, R5, R4H5 and R6 may be substituted one or several times, or may be unsubstituted. Preferred substituents are halogen and amino groups. Preferred substituted groups are 1,1,1-trifluoro-eth-2-oxy- and 1-bromo-eth-
It is a 2-oxy group. The alkoxy group is the group 0-(C)(2
-C'H2-0)n-R7, but in that case n
is an integer from 1 to 3, R7 is hydrogen, and the number of C atoms is 1 to 3.
It represents an alkyl group having 4, preferably a methyl group or a compound having 4 alkyl groups, preferably a methyl group. The alkyl radicals of the radicals R', R4R' and R6 are preferably hydrocarbon radicals having 1 to 4 carbon atoms, the methyl radical being particularly preferred.

The aralkoxy radicals R2R3R4RIS and R6 preferably have 1 to 4 carbon atoms in the alkyl part and 6 to 10 carbon atoms in the aryl part. Particular preference is given to the benzyloxy group.

The radicals R'' R3R4Ra and R6 are fluorine, chlorine, bromine and iodine, with preference given to chlorine.

The alkyl group of the alkylcal, +f:nyl group preferably has 1 to 4 carbon atoms. The alkoxycarbonyl radical preferably has 1 to 4 carbon atoms.

The amino groups of the groups R2R3R4H6 and R6 and the carbamoyl group may be substituted one or two times or may be unsubstituted.

Substituents are preferably acyl groups and substituted or unsubstituted alkyl groups. Particularly advantageous as the acyl group is an acetyl group.

The glycosyl group of group X is a hequinose-viranone-based heptanolarye and polysaccharide group. Preference is given to monosaccharide groups. Glucoside and lachitoside groups are preferred as hexopyranosides. Among these, β-glycosides have proven to be particularly suitable here as well.

The acyl group of radical X preferably has 1 to 20 carbon atoms and may be saturated or unsaturated, @-domain or branched. Particular preference is given to the acetyl group. An aminoacyl group also belongs to the acyl group. Preference is given to groups as aminoacyl radicals which are linked via a carphocycl group of a naturally occurring α-monoamino acid. In this case, free polar groups of the amino acids, for example amino groups, may be protected by suitable protecting groups. A particularly preferred amino acid group is an alanyl group containing a tosyl group in the amino group.

The phosphate group is the group -Po3H2 and its salts. Cations in these salts include all positively charged ions. Preference is given to alkali metal, alkaline earth metal and ammonium ions; particular preference is given to sodium-potassium-magnesium and calcium ions.

When R2 and R3 are closed in one ring system, compounds are preferred in which the oxygen atoms or amine groups of the groups R2 and R3 are bonded to form a total six-membered ring. Preferred rings formed in this way are especially 2,2-dimethyl-1
, 3-dioxy ring and 2,2-dimethyl-5-oxazine ring.

If the radicals R3 and R4 or R5 and R6 are closed in a ring system, preference is given to aromatic systems as the ring system. Particular preference is given to benz-fused compounds.

If the radicals R4 and R6 are closed in a ring system, preference is given to ring systems having 5 to 6 carbon atoms.

Compounds of formula I are compounds of the formula ■: RJ R4 [wherein R1 to R6 represent the above] compounds of the formula ■N-Y [wherein X represents the above and Y represents a reactive group] ]
It can be produced by reacting with a compound of

If X is a glycosyl group, methods should be used that prevent oxidation of the strongly oxidation-sensitive aglycone component H during the reaction. Classical methods, such as the Königs-Knorr-Rea reaction (Ki: inigs-Knorr-Rea) using silver salts or oxidizing heavy metal salts as reaction components;
ktion) or similar methods derived therefrom,
It cannot be used in this case. This is because the naphthol derivatives used can be readily oxidized to dyes.

The naphthol of the formula ``is reacted with the activated sugar of 2 in an aqueous aqueous solution of total hydroxide.
leophilegruppe), e.g.
7, especially fluorine, chlorine or bromine, or sulfonyl oxy7
Compounds in which the radical 0 is toluenesulfonyloxy or hamethanesulfonyloxy T5 have proved particularly advantageous. In this case, it has proven advantageous to keep the hydroxy or naphthol group of the sugar in circulation during the amino group reaction by means of a suitable holder group.

If X is an acyl group, methods known to Ichimaru can be used for preparing esters from alcohols as well as special phenols by reaction with organic carboxylic acids. Prior to esterification, the functional groups of the amino acids are advantageously masked by suitable absorbing groups. After the reaction, the protection fund can be detached again. The reactive group Y is preferably a cleavable nucleophilic group. Particular preference is given here to halons, alcoholades and carboxylates. For example, this type of production is carried out by Liebigs AnIL Che.
m, pp. 319-339 and European Patent (gP-B) No. 0
No. 039880.

In order to prepare all compounds of formula I in which Particularly preferred are compounds of the formula (2) in which gold represents iodine) and Y represents a radical halide, preferably chlorine, bromine or iodine or an alcoholade. Particular preference is given to phosphorus oxytrichloride as the compound X--Y. a of the compound of formula ■
It is advantageous to shield the leaving amino group by a holding group which is capable of leaving 5T after the reaction.

A compound in which X represents -POZ2 or -pz can be converted into a phosphate by hydrolyzing it by a known method.

This salt of phosphoric acid can be prepared from the acid by inverse lambda ion exchange in a known manner.

Compounds of formulas (1) and (2) are known compounds or can be produced in analogy to known methods of organic synthesis.
ebigs Ann, Chem, Volume 519 (198
4), 2420 (1985), 1847 (1985),
251 (1985), 251 (1985), 140 (
1980), 297 (1987), 1321 (19
80), Zeitschrift Fair Natoufolschung
turforschung) Volume 40b, page 534 (
(1985), Vol. 41b, 377 (198, 15)]. In particular, the Journal Fair Practice
Hiemi (Journal fiir Praktis)
che Chemie) Volume 62, page 30 (1900
4-methoxy-1-naphthol is known from
This can be reacted by fermentation to form a blue colorant (so-called Lucilahis inci). This oxidation is known as analytical chemistry (Analytica
lChemistry ) No. ! Volume 16, page 2494 (1
It was used as a precautionary measure to detect peroxidase (1995).

Some 4-meth-1-naphthol derivatives as well as 4-
Methods for converting methoxy-1-naphthol into colorants by oxidation have been known for several years; has not previously been used as a hydrolytic enzyme substrate in a detection method (generating a charge).

Surprisingly, the new compounds of formula (1) according to the invention are very suitable as hydrolase substrates. These compounds are water-soluble, colorless, and stable unless they come into contact with hydrolytic enzymes. Very good water solubility is desired. If one or more of the radicals R3R4R5 and R6 in the formula
A compound of formula (1) representing H2-CH2-0)n-R' is used.

Compounds in which the water solubility of the substrate is significantly increased by the lipophilic group M41f can also be used, provided that the disadvantage of adding suitable organic solvents or detergents that increase the solubility again in order to reduce the polarity of the solvent in which the reaction is carried out is Can be used as a hydrolase substrate.

A hydrolase substrate is a compound that can be converted by a corresponding substance that has hydrolase activity. Two types of products are produced upon absorption of water from the substrate. The reaction then follows methods known from enzyme kinetics. It is a prerequisite that at least a large portion of the substrate is soluble in the bath liquid containing the substance with the hydrolase conjugate.

The hydrolase substrate in the method according to the present invention includes:
Particularly preferred are compounds of formula I in which the molecular moiety X is the same as the molecular moiety of the natural substrate of the corresponding hydrolase. That is, for example, when β- is a lactodase substrate, in the formula
A compound of the formula (1) in which β- is a lactosyto group is preferred, and as an esterase substrate, a compound of the formula I in which X is an acyl group is preferred; Certain compounds 2) are particularly preferred.

The hydrolytic enzyme conjugate is a substance that can be decomposed into the products of compound No. 2 with the use of water.

This includes enzyme group 3 of naturally occurring and artificially produced hydrolytic enzymes.

In particular, the method according to the invention proves suitable as a method for the total detection of glycosidases, preferably lactosidases and esterases, preferably lipases and phosphatases.

A substance having hydrolase activity is a compound of this hydrolytic element and another compound, such as an immunologically active compound or a nuclear acid. Immunologically active compounds include
Examples include habten, antigens, antibodies and immune complexes, including seven fragments of antibodies, such as Fab- or F(a
b') 2-fragments may also be mentioned. The combination of a hydrolase and this immunologically active compound is called a hydrolase-conjugate. The hydrolytic enzymes serve here to label the immunologically active compounds. The method according to the invention is particularly suitable for detecting immunologically active compounds using alkaline or resistant phosphatases or β-lactosidases as labels.

In this case, the sample is preferably a solid or liquid sample.

In the case of solid samples, they can be divided into soluble samples and mostly non-soluble samples.

Soluble samples are advantageously converted into liquid samples for detection.

The nearly insoluble sample r+ is a solid that has bound to its external or internal surface a substance having hydrolytic enzyme activity. The binding method is important in the method according to the invention <-1
What a theory. This bond may be, for example, covalent and ionic or the adsorption order may be biological:\j. Biological binding is the binding between biological binding partners such as biotin and avidin or streptavicin, which are used as antigens or haptens.

Is there a substance in which all hydrolase activity exists? The liquid sample to be detected is primarily an aqueous solution. These solutions may be, for example, solutions of hydrolytic enzymes or hydrolytic enzyme conjugates in water.

Mixtures such as salts, detergents, etc. are often added to these solutions, for example to increase storage stability. The method according to the invention can also be used in solutions of this type. The liquid sample may be a body fluid or a liquid obtained by addition or separation of components therefrom. This includes, for example, blood, blood plasma, serum or urine.

The liquid sample may advantageously be a liquid, such as occurs, for example, during the course of an immunological test procedure. For example, Annals Op Clinical Biochemistry Volume 16,
221 (1979) and advantageous modifications thereof, which are known to those skilled in the field of immunoassays. Substances with hydrolase activity can also be detected in this type of liquid using the method according to the invention. These solutions usually contain buffer substances, stabilizers, wetting agents, etc. that do not significantly interfere with the detection.

The concentration of the substance with hydrolase activity is advantageous.
0-' to 10-15 mol/l, preferably H10-'
It can be measured in the range of ~jQ-12 mol/l.

This method is suitable for sample solutions whose value is between 5 and 11, preferably between 6 and 10. The optimum value for carrying out the method depends on the hydrolase used.

In order to carry out the process rapidly, it is advantageous to operate at a value at or near the value of 11, at which the hydrolase has the highest activity. Outside this range, significant enzymatic hydrolysis may not occur or inactivation of the hydrolase may occur.

The detection reaction is advantageously carried out in an absorbent or swellable support.

Supports of this type are, for example, fleeces or films.

Preferably fleece. Fleece is a material with a paper-like structure consisting of #L males. Cellulose, plastic or mixtures thereof are preferably used as PR fiber material.

Also suitable are spongy and/or porous supports.

However, the detection reaction can also be carried out in any type of container, for example a cuvette. If the progress of the reaction is to be followed using an adsorption photometer, compounds of formula I are preferred which yield products that are soluble in the reaction medium. One method is that, depending on the polarity of the reaction medium, one or more of the groups R3R4RI5 and R6 are polar groups, such as carboxyl- or sulfo groups or groups of the formula -0(-0)I2-CH2-0)n-R The method is to use the compound 2, which represents . Another method for detecting substances with hydrolase activity in solution is to add solvents, such as organic solvents or detergents, which reduce the polarity of the reaction medium. By taking advantage of these additional components, the solubility of the hydrolytic enzyme product is increased.

The presence of an oxidizing agent is necessary to invent color during the indicator reaction. As oxidizing agent it is possible to use substances or mixtures of substances whose oxidizing power is above the value of free naphthol.

For example, potassium hexacyanoferato■, perboric acid [
/peroxidase, peroxide/peroxidase, tetrazo, lium salt or oxygen/bilirubin oxidase may prove particularly advantageous.

To carry out the method of the invention, the sample is mixed with the corresponding hydrolytic enzyme substrate and oxidizing agent.

Mixing the sample and the hydrolase substrate can be accomplished in a variety of ways.

The sample, the hydrolase substrate and the oxidizing agent may be mixed simultaneously or one after the other.

In the case of liquid samples, the hydrolase substrate or oxidizing agent may be added, for example, to a solution of a substance with hydrolase activity in the form of a solid, e.g. powder, tablet, lyophilizate, etc., or in the form of a solution of the sample. Can be done.

If the detection reaction is carried out in an absorbent or swellable support, it has proven particularly advantageous to place the sample on a support impregnated with the hydrolase substrate and optionally oxidizing agents and additives.

A solution of the substance mentioned above is prepared in the round to be impregnated, and the support material is impregnated with this solution. Subsequently, the impregnated support material is dried.

If the support is a film, the hydrolase substrate and the fermentation agents and additives are advantageously incorporated into the film during the entire production.

If the oxidizing agent is not present with the hydrolytic enzyme substrate, the oxidizing agent is mixed with the sample either before or after mixing the sample with the hydrolytic enzyme substrate.

If the detection reaction is carried out in a container, it has proven particularly advantageous to use the hydrolase substrate in solid form or in solution, optionally mixed with oxidizing agents and additives.

In this case too, the oxidizing agent or additive can be added separately.

In the case of solid samples, it may be advantageous to add the hydrolase substrate and/or the oxidizing agent to the sample in solution, for example if the substance with hydrolase activity is bound to the support material. However, it is also possible, for example, to first mix the ingredients and then add the liquid to produce a solution.

It is important for the results of the detection method that the hydrolase substrate is able to come into contact with the substance having hydrolase activity present in the sample so that an enzymatic reaction can occur.

A strong color develops during the indicator reaction, which is caused by tft
Depending on the type of change, the color ranges from red through the arm to turquoise color. The color intensity can be measured in known manner using a photometer, in particular a reflectance photometer. For this purpose, a light beam of a wavelength that can be absorbed by the product of the indicator reaction is irradiated. 55
Wavelengths from 0 to 750 nm, in particular from 600 to 700 nm, are preferred.

The color intensities of gold, obtained by determining the total concentration of substances with hydrolytic enzyme activity at known concentrations, are compared with the values of a calibration curve relating each intensity to a particular concentration. This comparison is preferably carried out using the total W, @.

Since color development rather than color change should be observed, evaluation can also be performed with the naked human eye. This has the advantage of not requiring the use of any equipment. Also, sources of error, such as incorrect operation of the device, are eliminated.

The method according to the invention can be used for the qualitative and quantitative detection of substances with hydrolase activity. For qualitative evaluation, observe whether coloring occurs or not. For periodic measurements, each color must have a specific l! The interlayer color F9. Visually compare the m gold and sound versions.

The method according to the invention can be used particularly advantageously for test supports. Advantages include, for example, that the method is simple, inexpensive, and gives real results. This is particularly reliable since no deletion phenomena occur on the test support, which is very advantageous.

A test support is a tool for detecting all substances by conducting a test. These generally consist primarily of a substrate or sheet on which the reagents required for the test are provided, often by means of a 7 lease or film.

Other coupling components, such as diazonium salts, are not used in the process according to the invention, since they cause side reactions or impair safety.

In immunological test methods, substances with hydrolase activity,
In particular, this also applies to samples that should be further investigated: i1r of the hydrolytic enzyme complex.

The method according to the invention can therefore be used in particular in immunological methods for detecting substances with hydrolase activity, for example for total analyte detection. Methods of this kind are known to experts in immunoassays [for example, Annale F. Ode Clinical Chemistry Volume 16, 2
21 pages (1979)]. This method can be modified in such a way that the desired detection of immunologically active compounds labeled with hydrolytic enzymes is carried out using the method according to the invention. For example, if the analyte is an antigen or a hapten, the following method is advantageous: Adding an excess of the conjugate consisting of the antibody against the analyte and the hydrolyzed J4 enzyme to the sample solution containing the analyte. Add. An immune complex consisting of the analyte and the conjugate is generated. Excess conjugate is bound by immunoreaction to a support to which excess analyte or analyte analog is bound. The solution containing the conjugate consisting of the hydrolase and the immune complex is separated from the total support and treated according to the method according to the invention. Experimental results for the hydrolase conjugate are obtained from which the presence of the analyte or tV can be inferred. Another method is the detection of a conjugate consisting of a hydrolase and antibody bound to a support. This is also possible according to the method according to the invention.

To a sample solution containing one analyte, an excess of a conjugate consisting of an antibody against the analyte and a hydrolase is added. An immune complex consisting of the analyte and the conjugate is generated. The excess conjugate is bound by an immunological reaction to the support to which other excess antibodies against the analyte are bound. The solution containing the excess conjugate from the hydrolase and the antibody is separated from the support and the conjugate of the immunoconjugate and the hydrolase bound to the support or contained in the solution according to the method according to the invention The conjugate between the antibody and the hydrolase is detected.

- Adding to the sample solution containing the analyte a known conjugate consisting of the analyte or analyte analog and a hydrolytic enzyme. This mixture is placed on a support to which antibodies to the analyte and analyte analog are bound in known deficiencies relative to the sum of analyte and conjugate.

A portion of the analyte and conjugate are bound to the support.

With the method according to the invention it is possible to detect the conjugate bound to the support or present in the solution after molecule i of the solution.

The same principle can be used when the analyte is an antibody, but in that case an antigen or an antibody corresponding to that antibody should be used instead of the antibody in the method.

The evaluation by the method according to the invention is carried out by detecting the hydrolytic enzyme conjugate with a preliminary immunological method for detecting the analyte. That is, from the presence of hydrolytic enzyme conjugates or in the quantitative assessment of the amount of hydrolytic enzyme conjugates detected,
The presence or presence of analyte in the sample originally used can be estimated. This can be done, for example, using a calibration curve.

Furthermore, the method according to the invention for detecting substances with hydrolase activity can be used in methods for detecting nucleic acids. Methods of this type are known to experts in the field of nucleic acid hybridization testing. In this test, single- or double-stranded nucleic acids, in particular DNA or RNA or fragments thereof, are detected, usually in combination with a hydrolase as an enzyme label, which is a measure of the concentration of the nucleic acid to be detected.

Detection of nucleic acids labeled with this hydrolase is advantageously carried out by the method according to the invention for detecting substances with hydrolase activity. The advantages of this new immunological method and method for the detection of nucleic acids result from the advantages of the method according to the invention for detecting substances with hydrolase activity.

Another object of the present invention is a drug for detecting all substances having hydrolase activity.
A compound with the formula ■ of the species′! ! i- Contains. Agents of this type are suitable for detecting substances with hydrolytic enzyme conjugates in a sample, with the addition of an oxidizing agent in a previous, simultaneous or subsequent step.

Preference is given to detection agents for substances with hydrolase activity which contain at least one compound of formula I and an oxidizing agent.

Furthermore, the drug contains additives which are not necessary for the actual detection reaction, but which produce beneficial effects. To this belongs a special monobuffer that makes it possible to carry out the detection in a constant singleton. This also includes wetting agents and detergents that uniformly wet the support material.

The medicament according to the invention advantageously has one or several absorbent or expandable supports. Supports of this type are, for example, fleeces or films, preferably fleeces. Also suitable are spongy or porous supports.

The medicament advantageously comprises one or several absorbent or swellable supports, on which the hydrolytic enzyme substrate and the oxidizing agent are impregnated together or separately, or in the form of a film. In some cases, it has been introduced. This drug can be produced by a known method.

The medicaments according to the invention can, for example, contain, in addition to the hydrolytic enzyme substrate and the oxidizing agent and any additives present, a solvent. A preferred solvent is water. In this case too, the hydrolase substrate and the oxidizing agent may be present together or separately in solution.

The medicament according to the invention may be in the form of a single powder or several powders or powder mixtures. In addition to the hydrolytic enzyme substrate and the oxidizing agent, this type of agent can also contain all the conventional inert, soluble optical fillers such as polyvinylpyrolene, polyethylene glycol, and the like. The powder or powder mixture can also be compressed into tablets.

This type of drug is most suitable for use in methods for detecting substances having hydrolase activity.

In order to detect all compounds in which β- has lactosidase activity, a compound of formula (2) in which X' is a lactosyto group is particularly suitable. These compounds are new.

Therefore, the formula lv: R3N2 [In the formula, RI R2R'R' R5 and R6 represent all of the above, and X' is β-represents a lactosyto group]
The compounds are also objects of the present invention.

This compound is distinguished by the fact that it can be advantageously used in the method according to the invention for detecting substances with β-lactosidase activity, since β-lactosidase substrates are relevant.

Another object of the present invention is a method for producing a compound of formula (1), which is a compound of formula (II): [wherein R1 to R6 represent the above-mentioned] compound of formula V: x'-Y CV) [ In the formula, X' is characterized in that it is reacted with a compound in which β-represents a lactosyl group and Y represents a reactive group, optionally using a protective group.

Another object of the present invention is to form a compound of the formula ■: R3R2 [wherein R1 represents hydrogen or an alkoxy group, R2 represents hydrogen, halobin, aralkoxy- or amino-group in alcohol, and R'R'Rδ and R6 are hydrogen, haloke9, alkyl-alkoxy-aralkoxy-car♂xyl-alkylcarvinyl-alkoxycarvinyl-carbamoyl-sulfo-
or an amino group km, where X represents a glycosyl phosphate or an acyl group, and its crystallization R2 and N3 R
3 and R4, R4 and R6, and Rδ and R6 may each be closed to form a -ri ring system, at least one of the groups R1 and R2 is represented by water ffi', and at least one of the groups RIR2 and R3 is is not hydrogen] in the above method for detecting a compound having total hydrolase activity.

Next, the present invention will be explained in detail with reference to examples.

Example 1 a) 4-methoxy-1-naphthyl-2,3,4゜6
-tetra-0-acetyl-β-D- is lactopyranoside acetobrome is lactose [1-brome-2゜5.4.
6-Chitler ○-acetyl-α-D is lactobira nose, full force (FLuka) ) 96-19 (253,
<S mmol) is dissolved in 200 ml of acetone and deoxygenated by introducing N2. The solution was heated under constant stirring and first dissolved in 14.3 μl of potassium hydroxide in 13 μl of water.
9 (254,9 mmol), acetone 2
00-4-methoxy-1-naphtho-/l/ (Li
ebigs Ann.

Chem, 140 (1980)) 18-5,
! A solution of i' (10 6 -2 mmol) is added dropwise within 10 minutes in each case.

The reaction mixture should be constantly boiling under a stream of 'J# during the dropwise addition. Continue stirring at the same temperature for another 4 hours.
Allow to cool, separate insoluble material, and concentrate the filtrate in high-X air. 3 mouthfuls of syrupy residue 100,114 yen each
and used as a crude product in the next synthesis step without further purification.

WIR chromatography (silica dal 60, soluble ffl
Agent Chloroform/ethyl acetate 20:1) RF-0
, 40 b) 4-Methoxy-1-naphthyl-β-D- is added to a suspension of the compound from Example 1 a) in anhydrous methanol 150111/0 in anhydrous methanol within 1 hour under wet orifice drainage. .5M sodium methanolade solution 25ml 1'
The fc reaction mixture is added so that the value remains around 13. After completion of the reaction (DC adjustment) the product is isolated by column chromatography using silica vir.

Collection: 10g Thin layer chromatography (Silical 60, eluent
Chloroform/methanol 3:1) R sea 0.40 IH-NMR (DMSO-d,): δ (ppm) =
3.41-3.80 (m.

6H); 3.92 (s, 3H); 4.52-4.
89 Cm, 4H'): 5-31(d, J =
51 (z, IH)y 6-84 (d, J -8,
5Hz, 1H): 7.16 (d, J-8-5Hz
-1H): 7-47-7.60 (m, 2H): 8-
08-8-17 (m, IHG 8.31-8.41 (
m.

1H).

MS (pos, FAB): rrV/e -3!
17 Example 2 b) 4-Chlor-1-naphthyl-β-D- is prepared from lactopyranosylated by 4-chloro-1-naphthol (Aldrich) and acetobrome from lactose in a similar manner to the method from Examples 1 a) and 1 b). 4-Chlor-1-naphthyl-β-ri produced lactopyranoside.

Thin layer chromatography (silica'fk60.flJ release agent chloroform/methanol 7:3) RF -0,5
7 1H-NMR (DMSO-d,): δ (ppm) =
3.30-3.95 (m.

10H); 5.05 (d, , T -5Hz, 1
H) 17.24 (d, J-8,5Hz# IH);
7.61-7.85 (mp 3H): 8.15-
8.22 (m, fH); 8-40-8-53 (ol
Ip fH) Example 3 4-propoxy-1-naphthyl-β-D-ff lactopyranoside 4-globoΦcy-1-naphthol [LiebigsAn
Chem, 140 (1980)] and acetoderom were prepared from lactose to 4-chloro-1-naphthyl-β in a manner analogous to the method from Examples 1 a) and 1 b).
-D- is lactopyranoside e! ! ! I left it behind.

Thin layer chromatography (Silicavir 60. Eluent
Chloroform/methanol 3:1) R1-0,42 13C-NMR (CD30D): δ(1)I)m) -
11,09125,78゜62.44.70.30.7
1.02.72.55.75.07.76.94°10
4.28.105.39.111.52.122.65
．． 123.27゜128.45.125.80.127
．． 61.128.43.148.51゜151.57 MS ((TM8), -derivative): rrV'e -
652 Example 4 Lactopyranoside 1-naphthyl-β-D- in 4-isopropoxy-1-naphthol and acetobrome were prepared from lactose in a manner analogous to the method from Examples 1 a) and 1 t)). Propoxy-1-naphthyl-β-
Riichi produced lactopyranoside.

Thin layer chromatography (silica dal 60, solution [+1
Chloroform/methanol! l:1)RF-0,5
3 MS (neg, FAB): m/e -
! + 63 Example 5 1-naphthyl-β-D- in 4-benzyl is lactopyranoside 4-benzyloxy-1-naphthol (T, iebig
Ann, Chem, 140 (1980)) and acetobrome from lactose Examples 1a) and 1b
) 4-benzyloxy-1-
Naphthyl-β-Riichi produced lactopyranoside.

Thin layer chromatography (silica 2 60, eluent chloroform/methanol 3:1) RF-0,46 MS ((TMS) 4-ill conductor): rV'e
= 700 examples 6 4-Trifluoroethoxy-1-naphthyl-β-D- is lactopyranoside 4-trifluoroethoxy-1-naphthol
), 534 (1985)] and acetopro was prepared from lactose by analogy to the method from Examples 1 a) and 1 b).
- produced lactopyranoside.

HRG chromatography (silicavir 60, eluent
Chloroform/methanol filtration: 1) RF -0,42 1H-NMR (CD30D): δ (ppm) -5-4
6-3-95 (m, 6T(): 4-57 (q, J
-9Hz, 2H): 4-86 (d, J -6)(
Z, IH); 6.81 (d, J -8Hz,
IH); 7.09 (d.

J”” 8 Hz, IH) * 7.38-7.48 (
m, 2H); 7-99-8.09 (m+ 1)I);
8.25-8.35 (m, 1H) Example 7 2-Methoxy-1-naphthyl-dihydrobinphosphate Content 500 ml with stirrer, m dark and dropping funnel
2-methoxy-1-naphthol in 150μ of pyridine in a three-necked flask [Journal Ode de Chemical
Society (Journal of the
Chetnical 5ociety) 23.1
6 (1967) 115.079 (0.075 mol) phosphorus oxytrichloride 11.59 (6.9rnl, 0.075 mol) at 0-5°C under cooling.
mol) was gradually added dropwise under stirring. After 2 hours, the residue was aspirated and a! The liquid was poured onto 200 y of ice.

Adjust to 78% with IJ solution (concentrated with hydroxide under stirring for 5 minutes) and concentrate the yellow solution in vacuo. The light-colored residue is stirred with acetone and filtered with suction. Boil the crude product in boiling water 2
Dissolve the insoluble components in 0.00% alcohol, filter with suction, add concentrated hydrochloric acid to the mother liquor until the phosphate ester precipitates, filter with suction, and dry the reaction product over diphosphorus pentoxide until the image quality is constant. . 16 g of 2-methoxy-1-naphthyl-dihydrobinphosphate are obtained as colorless crystals.

Melting point: 171°0, "S'Iv'e = 254.1
8 Thin layer chromatography [Silica sol 60-Merck (
Merck), eluent: isopropanol/dityl acetate/H2O (5D: 50: 20)]
: RF-0,38 ``H-1(MR(D20.Na0D)'δ(ρp
rn) -5,98(s,3H)t 7-42 (dd
d, J-8-2-6, 8und 1.5 Hz, IH
): 7.46 (d, J -8-8Hz.

1H); 7-55 (tXdde J-8-2+ 6
-8 and 1.5 Hz II (); 7.71
(a, , T -9, OHz, IH); 7.86
(a, J-=18.2 Hz, IH); 8.3-
6 ppm (d. 15.55 g of phosphorus oxytrichloride (
9,16d) Add t-drops.

After stirring for 2 hours, the crystalline residue is filtered with suction, and the orange-colored filtrate is added dropwise to ice water 4001. P) by dropwise addition of concentrated sodium hydroxide solution using a lath electrode and measurement P1 (8 to 9 NIL) and evaporated to dryness in vacuo at 50° C. on a rotary evaporator.

18 g of a brown resin is obtained; this is dissolved hot in 100 d of isozoro 7 manol, dimethyl ether 3oOrI
The product is precipitated by addition of Lt. 4-Methoxy-1-naphthylphosphate-disodium salt 16.8
g is obtained.

"H-N+lF'= (D20+ Na0D) : δ(
ppm) -4-02 (s, 3H): 6-98 (d
# J = 8.5Hz, IH)y 7-58 (d
d, J = 8.5 and 1.5 Hz, IH
) near, 61.8.19 and 8.51 ppm
(ABYZ-system, 2 and each IH) Example 9 1-Acetoxy-4-methoxynaphthalene 4-methoxy-1-naphthol (1,75 &, 10 mmol) in acetic anhydride (18 rnt) under cold flow for 2.5 hours Bring to a boil. Thereafter, the solution is concentrated in vacuo and the oily residue is purified by column chromatography (silica vir; toluene/acetic acid ester = 40/1). After concentrating the corresponding fractions, the crude total petroleum ether (boiling point 90-1
Recrystallize from 10°C; yield 1.27.9 (5
8); Colorless crystals, melting point 51-53°C.

Example 10 Detection of human chorionic punadotrobin (hcG) in a sample A) Manufacture of test support 1 (Figure 1) a) Manufacture of fleece 3
) Model 42101e strip (e12.6 cm, width 0
．． <5CIn). 4-Methoxy-1-naphthyl-β-D- in DMSO is lactopyranoside 40m9
To 20 μt of a solution of /rnl was added 20 μt of a polyvinyl alcohol 24 width solution in water. This mixture was applied to the center of each piece of paper.

b) Manufacture of fleece 5 A piece of paper [Firma Kalff 7]
) manufactured by Mold 4210)' into a strip (length 2.6 cm, width 0.
6c7n). One end i PBS-buffer (
It was impregnated with 100 μt of phosphate buffered table water, pH 7, 0.1 Mibu serum aldemin, 0.1 Mibu Tween 20). In the center of the strip, a monoclonal antibody against hCG was added.
Conjugate consisting of ab-fragment and β-lactosidase (M that exhibits lactosidase activity) 20
U/ml, 100 μy/1 of seven clonal mouse antibodies against the β-chain of hen and 4-aminobenzyl-
1-thio-β-D- is lactopyranoside 85+ny/μ
7.5 μm of a solution containing . The end opposite to the buffer-impregnated end was impregnated with 10 μt of a 5e6 solution of polyvinyl alcohol in PAt-water.

C) Manufacture of fleece 6 A piece of paper [Schleicher & Juhl (FirmaSc)
hleicher & l 5chull ) Company 1! ! 3
512 type] was activated with bromcyan, and then a sheep antibody against the Fc region of the mouse antibody was immobilized. This piece of material was cut into strips (1.1 cm long and 0.6 cm wide).

d) Manufacture of the fleece 7 A sheet of paper [Whatmann
) Model D-28 made by Co., Ltd.] It was cut into a size of 5 ft x 0.6 ft.

e) The fleeces 2 and 3 are separated from each other by a plastic sheet 4.

The support 1 shown in FIG. 1 was produced by pasting the dried fleece onto a substrate sheet 2 having a width of 0.61 mm.

The fleece 5 is applied in such a way that the edge impregnated with polyvinyl alcohol points towards the edge 8 of the test support.

B) Test Act a) Sample Preparation Sample 0.5 d was added to a solution of 0.5 d of sodium perborate in phosphate buffer PI''! b) Application of the sample to the test strip The end 8 of the test strip 1 was placed in the solution.

A solution containing hC(') is introduced into Fleece 5 and Fleece 3 to dissolve the substances present therein.

hCG The beta-monoclonal antibody against hcG is converted into a five-component beta in solution in Fleece 5 by reaction with a lactosidase-labeled Fab-fragment and a monoclonal antibody against the beta-bond of hcG.
An immune complex in which - was labeled with lactosidase was formed. This immune complex, which in this case is a substance with hydrolase activity, is detected in Fleece 6 using the method according to the invention. The solution containing the immune complex penetrated from fleece 3 into fleece 6 just like the solution containing the substrate, where it was mixed with this substrate solution. In fleece 6,
The formed immune complexes as well as monoclonal antibodies against the excess β-chain were coupled to Fries 6 via immobilized sheep antibodies. An optional excess of β-lactosidase-labeled Fab fragments was further passed into Fleece 7. By lactosidase labeling and using an oxidizing agent, a blue color was formed in 7 Reese 6 within 10 minutes of the bound immune complex β-.

If the sample does not contain any hcG, β-
However, lactosidase-labeled immune complexes could not be produced. Substances with hydrolase activity are therefore not bound in the fleece 6 and therefore no color development is observed. In contrast, β-lactosidase-labeled Fab
- The fragments are contained in the total tt solution, which penetrates into the fleece 7. Therefore, β in fleece 7
Color uniformity was observed due to the reaction of the Fab-fragment labeled with lactosidase, which is also a substance with hydrolase activity. The test is thus controlled by carrying out the method according to the invention also in Fleece 7; this is particularly important if no hCG was present in the sample.

Evaluation can also be done quantitatively. It is h in the sample
This is because the more cfl and therefore the more β-lactosidase-labeled immune complex bound in fleece 6, the stronger the coloring will occur in fleece 6 after 10 minutes.

Example 11 Detection of Thyroxine (T4) A) Preparation of test strip 11 (FIG. 2) a) Fleece 14 Fleece 14 is impregnated with a conjugate of β-lactosidase and an antibody against T4.

b) Fleece 15 A piece of paper (Schleicher & Joule 3512) was activated with bromine cyanide and bound to it by T4 succinimide ester.

C) Fleece 16 Fleece 16 is 4-methoxy-1-naphthyl-β~D-
is a fleece impregnated with lactopyranoside.

d) 711-S 131d Useful for sample loading.

To produce the entire test support 11, the fleece was fixed onto a substrate sheet 12 as shown in FIG.

B) Conducting the test a) Sample preparation Samples were prepared in the same manner as fl+10.

b) Implementation The end 17t of the test strip 11 was placed in the solution.

The sample with T4 penetrated into fleece 13.

The sample then flowed further into Fleece 14, which separated the β-lactosidase complex.

The immunoreaction between the antibody and T4 produced an immune complex in which β- was identified as a lactondase ring. In addition to this immunoperitoneal association, a solution containing an excess of β-conjugate on Lactocedar percolated into the fleece 15. This excess was then bound to the paper via the bound T4.

Note that the solution containing the immune complex penetrated into the fleece 16. Therefore, β, a substance with hydrolase activity,
- The method according to the invention for the detection of total lactosidase-labeled immune complexes is carried out. The more this complex, and therefore also T4, is present in the solution, the more
After 0 minutes, Fleece 16 became much more strongly colored.

In the absence of T4, fleece 16 remained white lA.

[Brief explanation of the drawing]

FIG. 1 shows a test support according to Example 101C of the present invention. FIG. FIG. 2 is a test support according to Example 11 of the present invention. FIG. 1.11... Test support, 2... Substrate sheet, 4
...Plastic sheet, 3, 5, 6, 7°13.1
4, 15, 16...Fleece, 17゜18...End part -1-J 1.11 + Strap support 2, 12 Decal/-T Fig. 1 Horn' Cow, -t゛Rustic/ 8.17・End part Figure 2/〆

Claims (1)

[Claims] 1. A method for detecting a substance having hydrolase activity in a sample by mixing the sample with a hydrolase substrate and an oxidizing agent and evaluating the resulting color intensity, comprising: As an enzyme substrate, Formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1 represents hydrogen or an alkoxy group, and R^2
represents hydrogen, halogen, alkoxy-, aralkoxy- or amino-group, R^3, R^4, R^5 and R^6
may be the same or different, hydrogen, halogen,
represents an alkyl, alkoxy, aralkoxy, carboxyl, alkylcarbonyl, alkoxycarbonyl, carbamoyl, sulfo or amino group,
X represents a glycosyl, phosphate or acyl group, in which case the groups R^2 and R^3, R^3 and R^4 and R
^4 and R^5 and R^5 and R^6 may each be closed to form a ring system, at least one of the groups R^1 and R^2 represents hydrogen, and the group R^1 , at least one of R^2 and R^3 is not hydrogen]
A method for detecting a substance having hydrolase activity, the method comprising using seeds. 2. At least one compound of formula I
An agent for detecting a substance having hydrolase activity in a sample, characterized by containing a species. 5. Formula IV: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R^1 represents hydrogen or an alkoxy group, and R^2
represents hydrogen, halogen, alkoxy-, aralkoxy- or amino-group, R^3, R^4, R^5 and R^6
may be the same or different, hydrogen, halogen,
represents an alkyl-alkoxy, aralkoxy-, carboxyl-, alkylcarbonyl-, alkoxycarbonyl-, carbamoyl-, sulfo- or amino-group, X' represents a β-galactosyl group, in which case the groups R^2 and R^ 3.R^
3 and R^4, R^4 and R^5, and R^5 and R^6 may each be closed to form a ring system, and at least one of the groups R^1 and R^2 is hydrogen. represents the group R^1, R
At least one of ^2 and R^3 is not hydrogen].